This invention relates to simultaneously reacting multiple chemical samples using arrays. In particular, the invention relates to an apparatus for and a method of conducting multiple chemical reactions or assays simultaneously using microarray technology for diagnostic, therapeutic and analytical applications.
Devices for processing multiple biological assays are known (U.S. Pat. Nos. 5,545,531 and 6,083,763, for example). The devices typically comprise reaction vessels having a plurality of reaction wells, each well has sidewalls and a closed end or bottom and an array of chemical samples fixed to the closed end. The end opposite to the closed end is open to access the reaction well for performing the assays with the array. Conventional microtiter plates are examples of conventional reaction vessels.
One conventional reaction vessel is made from a biochip wafer having a plurality of biological arrays fixed to the wafer surface and a microtiter-type plate attached to the wafer surface. The microtiter-type plate has a plurality of through holes formed completely through the thickness of the plate instead of closed-end wells as described above. The through holes in the microtiter-type plate are aligned with the plurality of arrays on the wafer. Reaction wells are formed by the attachment, wherein the wafer becomes the closed end and the through hole walls are the sidewalls of each reaction well. The arrays on the wafer are accessible via the open ends. Biological test samples are added to the open end to perform multiple assays at one time.
Another conventional reaction vessel is a conventional type of microtiter plate having a plurality of wells, wherein each well has sidewalls and a closed end. Individual biochips are attached inside each well. The individual biochips are obtained by dicing up the biochip wafer mentioned above. The individual biochips comprise an array of chemical samples fixed to a chip surface. The chip is adhered to the closed end of the well, such that the array surface of the chip is accessible from the open end of the well. Again, biological test samples are added to the open end to perform multiple assays at one time.
U.S. Pat. No. 5,324,633 discloses still another conventional reaction vessel that utilizes a flow cell having reservoirs that align with arrays of reactants on a substrate when the substrate is placed on the flow cell. Channels extending between the reservoir and the outside of the flow cell are used to inject the test sample into the reservoirs after the reaction vessel is assembled. O-rings are used to seal the substrate to the flow cell around each reservoir. Alternatively, the reaction vessel is a channel block having multiple channels therein. The channel block is mated with a substrate supporting arrays of biological material. The channels of the channel block are then filled with different reaction solutions that react with the arrays of biological materials similar to the flow cell.
The conventional reaction vessels are not self-contained (e.g., gas tight or fluid tight). Therefore, special handling and processing equipment must be used to perform assays using these conventional reaction vessels in order to provide stable handling and to control assay conditions, such as atmosphere, temperature, contamination, and prevent loss of sample or reagents, and the like. The special handling and processing equipment is expensive and not conducive to increased throughput, without added expense. Moreover, additional materials and assembly steps are needed if one skilled in the art wanted to seal the conventional reaction vessels to render them gas or fluid tight. For example, an additional cover plate or film must be placed over the open ends of the plurality of wells of the conventional reaction vessel to close or seal the open ends after the assay ingredients are added to the array therein. In the flow cell embodiments, the channels must be blocked with a cap or plug, for example, to seal the reservoirs.
Thus, it would be advantageous to have a method of simultaneously conducting multiple chemical reactions and a reaction vessel that is fully enclosed and gas, liquid and/or fluid tight without the need for cover plates and/or caps and the associated additional assembly steps. Such an apparatus and a method could decrease cost and increase throughput in the art of performing multiple assays.
The present invention provides a novel method of simultaneously conducting multiple chemical reactions and a reaction assembly apparatus that are low cost because the present invention can use pre-existing equipment, materials and well-known techniques to provide a self-contained, gas, liquid and/or fluid tight reaction vessel. In particular, the present invention is an apparatus for and a method of simultaneously conducting assays of multiple biological samples, such as assays of proteins, nucleic acids, specific binding pairs, and the like, advantageously that can use conventional microarrays and microtiter plates in a low cost manner for diagnostic, therapeutic and analytical applications, for example. The method and apparatus of the present invention can be subjected to handling and reaction conditions that are not available to the conventional reaction vessels described above. The handling and reaction conditions promote or accelerate the chemical reactions within the apparatus.
In one aspect of the invention, a method of simultaneously conducting multiple chemical reactions in a reaction assembly that comprises a microtiter plate of wells containing test samples and a microarray of sets of chemical reactants is provided. The method comprises the step of assembling the microarray of sets of chemical reactants to the microtiter plate of test samples such that the microarray covers open ends in the test sample wells of the microtiter plate to form a plurality of closed cells. Each closed cell comprises a set of chemical reactants and a respective test sample. The method further comprises the steps of sealing the microtiter plate to the microarray to create one or more of a gas, a liquid and a fluid tight seal; and mechanically agitating the sealed reaction assembly to contact test samples with the chemical reactants in each closed cell simultaneously.
In another aspect of the invention, a method of simultaneously conducting multiple chemical reactions between a first chemical sample and a second chemical sample is provided. The method comprises the step of providing a plate, having a plurality of spatially arranged wells in a well array pattern. Each well has a side wall adjacent to a closed end that enclose the well except for an open end opposite the closed end. The open end is adjacent to the plate surface for receiving a volume of the first chemical sample. The method further comprises the step of providing the second chemical sample bound to a surface of a substrate in an array pattern of features. The array of second chemical samples is spatially arranged in sets of features on the substrate surface similar in layout to the arrangement of the wells in the plate.
The method yet still further comprises the step of assembling the array and the plate into a reaction assembly and sealing the assembly. The array and plate are assembled such that the array substrate encloses the open ends of the plurality of wells in the plate. The features of the second chemical samples on the array are aligned with the open ends of the wells in the plate. Such aligned features and wells become multiple separate closed cells or reaction chambers in the reaction assembly. The reaction assembly is sealed to have one or more of a gas, liquid and fluid tight seal between the plate and the array, such that each closed cell is sealed.
The method yet still further comprises the step of simultaneously contacting the first chemical sample with the second chemical sample for a period of time in the plurality of closed cells to facilitate reactions therebetween. A variety of reaction parameters may be used for accomplishing the chemical reactions in accordance with the invention. Advantageously, the reaction assembly may be inverted and/or mechanically agitated under controlled temperature conditions to cause mixing between the first and second chemical samples in each closed cell.
In another aspect of the invention, an apparatus that provides for simultaneously conducting multiple chemical reactions is provided. The apparatus is a reaction assembly that comprises a plate having a plurality of spatially arranged wells in a well array pattern. Each well has a side wall adjacent to a closed end that enclose the well except for an open end opposite the closed end. The open end is adjacent to the plate surface for receiving a first sample into the wells. The apparatus further comprises an array comprising sets of a second sample bound to a surface of an array substrate in an array pattern of features. The sets of second samples are spatially arranged on the substrate surface similar in layout to the arrangement of the wells in the plate. Advantageously in one or more embodiments, the plate can be a conventional microtiter plate and the array can be a conventional microarray. The array is assembled to the plate, such that the array surface faces the plate surface, and such that the features on the array surface are aligned with the open ends in the plate. The array substrate covers the open ends of the wells in the plate to form a plurality of closed cells. Each closed cell comprises a respective set of second samples and a respective first sample. The apparatus further comprises a seal between the array substrate and the plate to provide one or more of a gas tight, a liquid tight and a fluid tight seal.
For the method and apparatus, the seal may be a seal formed by any one of using a flexible (pliable) array substrate and applying pressure, using a rigid or a flexible array substrate and an adhesive, using a pliable gasket between the array substrate and the plate and applying pressure, using the pliable gasket and an adhesive, and using a pliable gasket integral with the surface of the plate and applying pressure and/or using an adhesive. Pressure may be applied by one or more of mechanical clamps, external fluid pressure and vacuum, for example. Where an adhesive is used, one or more of heat, pressure and radiation may be used, but may be removed after the adhesive is cured or otherwise adhered to the surfaces.
In still another aspect of the invention, a kit for simultaneously conducting multiple assays of biological materials is provided. The kit comprises one or more of a plate having a plurality of wells spatially arranged in a surface of the plate, each well having a closed end, an open end opposite the closed end, and a side wall between the closed end and the open end; an array having a plurality of sets of biological features attached to one surface of an array substrate in a spatial arrangement similar to the well arrangement; a pliable gasket having a plurality of spatially arranged through holes, where the spatial arrangement of the through holes is similar to the spatial arrangement of the wells and the sets of biological features, and where the gasket is used to seal together the array and the plate; an adhesive for sealing together at least the array and the plate; and a sample of biological material as a control. The kit may optionally further comprise instructions for simultaneously conducting multiple assays according to the method of the present invention using the assembled reaction assembly apparatus of the present invention.
The reaction products produced by any of the above methods in the reaction assembly apparatus can be analyzed in accordance with the invention using well-known techniques and equipment. In one embodiment, the reaction products can be analyzed with the introduction of radiation into each closed cell, for example, through the plate or through the array substrate of the reaction assembly. In this embodiment, at least one of the plate or array substrates is transparent to the radiation, such as electromagnetic, particle, or ultrasonic radiation, which may be used in the evaluation of the reaction products. In another embodiment, the reaction assembly is disassembled to access the reaction products on the surface of the second substrate or array. In this embodiment, the surface may be rinsed to remove any unreacted test sample and prevent further reactions from occurring, before evaluation. In this embodiment, the array substrate may be transparent to the radiation used for analysis, or it may reflect or absorb the radiation used for analysis.
The apparatus and method of the present invention advantageously provide a reaction assembly to perform simultaneous multiple chemical reactions that uses the array substrate to cap or close the open ends of the wells in the plate. The reaction assembly is sealed using the material characteristics of the array substrate, a gasket, pressure and/or an adhesive to provide one or more of a gas, a liquid, or fluid tight seal. No other materials, such as conventional covers or caps, or assembly steps are necessary for the invention.